Development of a vascular supply is a fundamental requirement for many physiological and pathological processes. Actively growing tissues such as embryos and tumors require adequate blood supply. They satisfy this need by producing pro-angiogenic factors, which promote new blood vessel formation via a process called angiogenesis. Vascular tube formation is a complex but orderly biological event involving all or many of the following steps: a) endothelial cells (ECs) proliferate from existing ECs or differentiate from progenitor cells; b) ECs migrate and coalesce to form cord-like structures; c) vascular cords then undergo tubulogenesis to form vessels with a central lumen; d) existing cords or vessels send out sprouts to form secondary vessels; e) primitive vascular plexus undergo further remodeling and reshaping; and f) peri-endothelial cells are recruited to encase the endothelial tubes, providing maintenance and modulatory functions to the vessels; such cells including pericytes for small capillaries, smooth muscle cells for larger vessels, and myocardial cells in the heart (Hanahan, D., Science, 1997, 277, 48; Hogan, B. L. & Kolodziej, P. A. Nature Reviews Genetics, 2002, 3, 513).
The development of the blood vessels is strictly controlled. Up to the present, a large number of secretory factors produced by peripheral cells are known to regulate differentiation, proliferation and migration of ECs and coalescence into cord-like structures. The angiogenesis-promoting factors reported thus far can be largely classified into a few groups. They are mostly growth-inducing factors that induce cellular growth, cytokines having immune activity, hormones, or lipid products (Bussolino F et al., Trends Biochem. Sci., 22(7), pp. 251-256, 1997).
Vascular endothelial growth factor (VEGF) has been identified as the key factor involved in stimulating angiogenesis and in inducing vascular permeability (Ferrara et al., Endocr. Rev. 18: 4-25, 1997). A form of murine VEGF gene was identified and the expression pattern was analyzed during its embryogenesis. Continued expression of VEGF was observed in spear-shaped epithelial cells neighboring the endothelium, e.g. the choroid plexus and the glomerulus of the kidney. These data correspond to the role of VEGF as a multifunctional factor regulating the growth and differentiation of the ECs (Breier, G. et al., Development, 1992, 114, 521).
VEGF is involved in angiogenesis in all connective tissues (e.g., lungs, heart, placenta and solid tumors) (Binetruy-Tourniere et al., EMBO J. 2000, 19, 1525). For example, VEGF is involved in growth of solid tumor and metastasis by stimulating tumor-related angiogenesis (Lu et al., J. Biol. Chem. 2003, 278, 43496). Also, since the VEGF expression is essential in the restoration of connective tissues, use of the VEGF for the restoration of the connective tissues was proposed.
Actually, Chen et al. disclosed a method for synergistically enhancing endothelial cell growth in an appropriate environment therefor which comprises adding to the environment, VEGF, effectors and serum-derived factor in U.S. Pat. No. 5,073,492 (Dec. 17, 1991). Also, vascular endothelial cell growth factor C subunit DNA is prepared by polymerase chain reaction techniques. The DNA encodes a protein that may exist as either a heterodimer or homodimer. The protein is a mammalian vascular endothelial cell mitogen and, as described in European Patent Application No. 92302750.2 (Sep. 30, 1992) is known to be useful for the promotion of vascular development and repair as it is.
Despite the advancement regarding VEGF, when subjected to animal experiments, it fails to pass through the blood barrier due to its large molecular size like other proteins and is eliminated in short time due to short half-life. Although studies are actively carried out to find out the important EC-specific genes involved in angiogenesis and thus to treat various angiogenesis-related diseases, there is no substantial result as yet.